The PTEN tumor suppressor (see WO98/34624 which is hereby incorporated by reference in its entirety) is a cytoplasmic phosphatase which dephosphorylates the important second messenger phosphatidylinositol 3,4,5-triphosphate (Maehama and Dixon 1998). This activity downregulates the many oncogenic signals initiated by PIP3 activation of Akt including anti-apoptotic pathways, cell cycle progression and increasing cell metabolism (Sulis and Parsons 2003). The role of PTEN in cancer is evident from its frequent loss, either genetically or functionally, in many different tumor types (Bonneau and Longy 2000). Originally discovered as deleted in glial cancers, it has since been implicated in tumorigenesis of the prostate, breast, endometrium, melanocytes, kidneys and lungs. Germline mutations in PTEN were also linked to inherited cancer predisposition syndromes such as Cowden's Syndrome (Eng 2003). Mouse models of PTEN loss have recapitulated its role as an tumor suppressor both in the heterozygous mouse and tissue specific knockouts in many different tissue types (Di Cristofano, Pesce et al. 1998; Kwabi-Addo, Giri et al. 2001; Petrocelli and Slingerland 2001; You, Castrillon et al. 2002; Fraser, Zhu et al. 2004).
The PTEN protein contains an N-terminal dual specificity phosphatase domain, and a C-terminal C2 phospholipid binding domain, followed by an unstructured tail of regulatory importance because of the phosphorylation sites found within (Lee, Yang et al. 1999; Vazquez, Ramaswamy et al. 2000; Torres and Pulido 2001; Vazquez, Grossman et al. 2001). PTEN protein is mostly cytoplasmic however there is increasing evidence for a PTEN presence in the nucleus, a localization which is regulated by the monoubiquitination of the protein by NEDD4-1 (Baker 2007; Wang, Trotman et al. 2007).
Ribosome scanning of the 5′UTR precedes translation initiation which occurs at the start codon, AUG. Though the actual means by which the ribosome decides the proper start codon remains incompletely understood, there are certain properties of both the mRNA itself and the sequence which will dictate where the pre-initiation complex will slow its scanning and start to translate. The classic “Kozak sequence” CCACCATGG, where the underlined ATG is the initiation codon, has been shown to be the most favorable sequence context for initiation (Kozak 1991). mRNA secondary structure also promotes initiation probably by an actual slowing of the scanning of the pre-initiation complex which requires a helicase to melt secondary structures prior to reading (Kozak 1990).
In certain transcripts, translation initiation can occur from non-AUG codons. This usually comprises only a minor percentage of the total protein translated from a transcript and the result is a mixed species of proteins varying at their N-termini. Kozak delineated the efficiencies of translation initiation from non-AUG codons and found that GUG and CUG were both capable of initiating translation in vitro however far less efficiently (Kozak 1989). Further research has shown that the availability of methionine can alter the promiscuity of translation initiation through a mechanism that remains unclear, but probably involves the phosphorylation of eIF2, a component of the 43S pre-initiation complex, by a nutrient sensitive kinase (Hershey 1991; Hann 1994).
A number of proteins have been shown to be translated from alternate initiation codons. The transcription factor, c-myc, has an alternate upstream CUG initiation codon which when translated, adds 14 amino acids to the N-terminus of the protein (Hann and Eisenman 1984). This alternate isoform has been shown to be selectively disrupted in Burkitt's lymphoma (Hann, King et al. 1988). In tissue culture the longer form of myc is predominantly transcribed at high cell densities when methionine is at a low concentration (Hann, Sloan-Brown et al. 1992). Further studies have revealed that the longer form of c-myc is growth inhibitory and has a different set of transcriptional targets than the classic c-myc protein (Hann, Dixit et al. 1994). (Florkiewicz and Sommer 1989) (Prats, Kaghad et al. 1989).
Additionally, it is known that the actual subcellular localization of a protein can be dictated by alternate initiation codons. In the case of the mouse proto-oncogene int-2 alternate initiation from an upstream CUG codon encodes a nuclear localization while the AUG codon encodes a signal peptide for localization to the secretory pathway (Acland, Dixon et al. 1990). A similar phenomenon was described in the human FGF3, in which the protein translated from AUG is destined for the secretory pathway while the protein translated from an upstream CUG is localized to the nucleus (Kiefer, Acland et al. 1994). Furthermore, in some eukaryotic proteins, such as TEF-1 and PRPS-3, the protein is completely initiated from a CUG codon (Taira, Iizasa et al. 1990; Xiao, Davidson et al. 1991).
Proteins that are destined for secretion are targeted to the endoplasmic reticulum by a stretch of hydrophobic amino acids called a signal peptide (Blobel, Walter et al. 1979). Usually found at the N-termini of proteins, the signal peptide binds the signal recognition particle (SRP) upon translation and causes the ribosome to halt and translocate to the rough endoplasmic reticulum where it binds the SRP receptor. Once the ribosome docks, the SRP-SRP receptor complex is released and translation resumes through the lumen of the ER through the Sec61 translocon. The signal peptide is then cleaved off in the case of soluble proteins releasing the protein from the Sec translocon. In the case of proteins spanning a membrane, the transmembrane helix serves as a signal peptide for ER translocation. These proteins are modified extensively by glycosylation in the golgi and are shuttled to the plasma membrane in secretory vesicles (Alberts 2002).
There are a number of secreted proteins that have been shown to be important in cancer. The Wnt signaling pathway for example has been shown to be altered in lung cancer. Wnt is a secreted ligand for the family of Frizzled receptors. Wnt activation of frizzled causes disheveled to dissociate the β-catenin degradation complex, which includes APC, allowing for levels of β-catenin to rise and translocate to the nucleus where it can interact and transactivate the TCF transcription factor. Inactivating mutations in APC and activating mutations in β-catenin have been detailed in both inherited and sporadic colon cancer. Additionally, a number of extracellular ligand antagonists such as SFRP and Wnt-5a compete for the same Frizzled receptors as Wnt. Both have been shown to be tumor suppressors; the SFRP knockout mouse develops lymphoid tumors and epigenetic silencing of Wnt-5a has been detected in melanomas.
All published reports of PTEN have indicated that the protein is located in either the cytoplasm or nucleus. A number of extracellular proteins (glypicans and syndecans) were found to bind PTEN, as did a number of proteins involved in the secretory pathway (reticulocalbin and calumenin). It was assumed that PTEN entered the secretory pathway to allow for such interactions. In fact, as disclosed herein, a novel differentially translated protein exists, named PTEN-long, which contains an N-terminal signal peptide and which is secreted extracellularly.